Crystallization (Comment) | Organism |
---|---|
purified recombinant MOMT5 and MOMT9 enzyme variants in complex with S-adenosyl-L-homocysteine (SAH), coniferyl alcohol, or sinapyl alcohol, hanging drop vapor diffusion method, crystallization from 22% (w/v) PEG 4000, 0.3 M Mg(NO3)2, pH 7.2, 1 mM DTT, 1 mM SAH, and 1 mM monolignols, X-ray diffraction structure determination and analysis at 1.60-1.73 A resolution, modeling | Clarkia breweri |
Protein Variants | Comment | Organism |
---|---|---|
additional information | a number of monolignol 4-O-methyltransferase variants (MOMTs) are evolved that can trans-methylate the para-hydroxyls of monolignols, which prevents the incorporation of modified precursors into the lignin polymer by disrupting oxidative radical formation. The engineered enzyme is biochemically active against both coniferyl and sinapyl alcohol with similar catalytic turnover rates (e.g. MOMT3 and 4). Therefore, the transgenic plants with overexpressed MOMT maintained similar monomer composition and lignin structure as the wild-type plants | Clarkia breweri |
T133L/E165I/F175I/F166W/H169F | a promiscuous O-methyltransferase is incrementally evolved to constrain its substrate specificity to guaiacyl lignin precursors. The enzyme with nine amino acid substitutions is functionally specialized for selectively methylating the condensed lignin precursors. Specific remodeling the active site of a monolignol 4-O-methyltransferase creates an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. The engineered promiscuous enzyme MOMT5 is built by remodelling of its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors | Clarkia breweri |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0114 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT5 | Clarkia breweri | |
0.0147 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT5 | Clarkia breweri | |
0.0161 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT8 | Clarkia breweri | |
0.0161 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT9 | Clarkia breweri | |
0.0263 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT8 | Clarkia breweri | |
0.0681 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT4 | Clarkia breweri | |
0.1095 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT9 | Clarkia breweri | |
0.1187 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT3 | Clarkia breweri | |
0.1926 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT4 | Clarkia breweri | |
0.1985 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT3 | Clarkia breweri |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Clarkia breweri | O04385 | - |
- |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.0017 | - |
enzyme variant MOMT4, pH 7.5, 30°C, substrate caffeyl alcohol | Clarkia breweri |
0.0025 | - |
enzyme variant MOMT8, pH 7.5, 30°C, substrate coumaryl alcohol | Clarkia breweri |
0.0037 | - |
enzyme variant MOMT9, pH 7.5, 30°C, substrate coumaryl alcohol | Clarkia breweri |
0.0055 | - |
enzyme variant MOMT4, pH 7.5, 30°C, substrate coumaryl alcohol | Clarkia breweri |
0.0065 | - |
enzyme variant MOMT5, pH 7.5, 30°C, substrate caffeyl alcohol | Clarkia breweri |
0.0077 | - |
enzyme variant MOMT8, pH 7.5, 30°C, substrate sinapyl alcohol | Clarkia breweri |
0.0096 | - |
enzyme variant MOMT5, pH 7.5, 30°C, substrate coumaryl alcohol | Clarkia breweri |
0.0098 | - |
enzyme variant MOMT9, pH 7.5, 30°C, substrate sinapyl alcohol | Clarkia breweri |
0.0108 | - |
enzyme variant MOMT8, pH 7.5, 30°C, substrate caffeyl alcohol | Clarkia breweri |
0.0129 | - |
enzyme variant MOMT9, pH 7.5, 30°C, substrate caffeyl alcohol | Clarkia breweri |
0.0396 | - |
enzyme variant MOMT5, pH 7.5, 30°C, substrate sinapyl alcohol | Clarkia breweri |
0.0514 | - |
enzyme variant MOMT8, pH 7.5, 30°C, substrate coniferyl alcohol | Clarkia breweri |
0.0668 | - |
enzyme variant MOMT4, pH 7.5, 30°C, substrate coniferyl alcohol | Clarkia breweri |
0.0683 | - |
enzyme variant MOMT9, pH 7.5, 30°C, substrate coniferyl alcohol | Clarkia breweri |
0.0796 | - |
enzyme variant MOMT4, pH 7.5, 30°C, substrate sinapyl alcohol | Clarkia breweri |
0.0806 | - |
enzyme variant MOMT5, pH 7.5, 30°C, substrate coniferyl alcohol | Clarkia breweri |
0.1929 | - |
enzyme variant MOMT3, pH 7.5, 30°C, substrate sinapyl alcohol | Clarkia breweri |
0.3463 | - |
enzyme variant MOMT3, pH 7.5, 30°C, substrate coniferyl alcohol | Clarkia breweri |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | substrate specificities of engineered monolignol 4-O-methyltransferases, MOMT3, MOMT4, MOMT5, MOMT8, and MOMT9 | Clarkia breweri | ? | - |
- |
|
S-adenosyl-L-methionine + caffeyl alcohol | - |
Clarkia breweri | S-adenosyl-L-homocysteine + 4-methoxycaffeyl alcohol | - |
? | |
S-adenosyl-L-methionine + coniferyl alcohol | - |
Clarkia breweri | S-adenosyl-L-homocysteine + 4-methoxyconiferyl alcohol | - |
? | |
S-adenosyl-L-methionine + coumaryl alcohol | - |
Clarkia breweri | S-adenosyl-L-homocysteine + 4-methoxycoumaryl alcohol | - |
? | |
S-adenosyl-L-methionine + monolignol | - |
Clarkia breweri | S-adenosyl-L-homocysteine + 4-O-methylmonolignol | - |
? | |
S-adenosyl-L-methionine + sinapyl alcohol | - |
Clarkia breweri | S-adenosyl-L-homocysteine + 4-methoxysinapyl alcohol | - |
? |
Synonyms | Comment | Organism |
---|---|---|
MOMT5 | - |
Clarkia breweri |
monolignol 4-O-methyltransferase | - |
Clarkia breweri |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Clarkia breweri |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.006 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT8 | Clarkia breweri | |
0.017 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT9 | Clarkia breweri | |
0.033 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT5 | Clarkia breweri | |
0.049 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT8 | Clarkia breweri | |
0.063 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT9 | Clarkia breweri | |
0.069 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT5 | Clarkia breweri | |
0.173 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT3 | Clarkia breweri | |
0.247 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT3 | Clarkia breweri | |
0.271 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT4 | Clarkia breweri | |
0.528 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT4 | Clarkia breweri |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Clarkia breweri |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | - |
Clarkia breweri |
General Information | Comment | Organism |
---|---|---|
additional information | homology modeling and substrate docking analysis of enzyme mutant MOMT8, substrate binding in crystal structures of MOMTs, overview | Clarkia breweri |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.16 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT9 | Clarkia breweri | |
0.23 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT8 | Clarkia breweri | |
1.24 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT3 | Clarkia breweri | |
1.46 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT3 | Clarkia breweri | |
2.25 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT5 | Clarkia breweri | |
2.74 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT4 | Clarkia breweri | |
3.04 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT8 | Clarkia breweri | |
3.91 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT9 | Clarkia breweri | |
3.98 | - |
sinapyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT4 | Clarkia breweri | |
6.05 | - |
coniferyl alcohol | pH 7.5, 30°C, recombinant enzyme mutant MOMT5 | Clarkia breweri |